**4. Transgenic approaches for crop improvement**

Transgenic technology is the only technology through which alien genes may be introduced across the species. Sugarcane has well be explored to engineer for certain valuable agronomic traits and for enhanced sucrose contents. Most of the transformation events reported in sugarcane are through biolistic, anyhow *Agrobacterium* and electroporation have also been used. Success of engineered lines depend upon the integration and stable expression of introduced gene/s. Recalcitrancy, low transformation efficiency, transgene inactivation and difficult backcrossing are major bottlenecks in sugarcane transformation. Though limited reports are available for the field plantation of transgenic sugarcane yet a large number of research groups are involved in engineering sugarcane genome [58].

### **4.1. Developing genetically modified varieties with improved biotic stress tolerance**

#### *4.1.1. Herbicide resistance*

Herbicide resistance is one of the major traits in transgenic plants. It is so desirable that more than 70% of the transgenic crops growing worldwide are herbicide resistant. Various herbicide resistance genes i.e. *EPSPS*, *bar*, *aroA and BXN* have been have been transformed to crop plants for developing herbicide tolerant crops. Crops resistant to glyphosate and glufosinate have been cultivated since 1990s. Plants having ability to tolerate high dose of glyphosate have been developed through biolistic transformation whereas *Agrobacterium* and other methods of transformation (electroporation) have also been tested [59]. Gallo-Meagher and Irvine [60] reported *bar* gene transformation in sugarcane by biolistic gun. Resultant transformants showed tolerance against basta which authenticated effectiveness of *bar* gene not only for herbicide tolerance trait but also as a selectable marker gene, for the selection of putative transformants. Enriquez-Obregon et al. [61] reported transformation of *uid1* and *bar* genes in sugarcane by *Agrobacterium* mediated transformation. They obtained GUS-positive and BASTA resistant calli. Similarly, BASTA resistant variety (Ja 60-5) of sugarcane has also been developed through *Agrobacterium* mediated transformation. Transformation frequencies reached up to 10–35% by employing different transformation protocols. The *PAT* (phosphinothricin acetyltransferase) and *neo* (neomycin phosphotransferase) genes were reported to be transformed in SP80-180 genotype of sugarcane by biolistic method [62]. The selected transformants were resistant against commercial formulation of ammonium glufosinate. Southern blot analysis was used to confirm the stable integration of *neo* and *PAT* genes. While western blot analysis and RT-qPCR were used to analyze the expression of these genes. Another report was given by Manickavasagam et al. [5]. They developed herbicide tolerant sugarcane plants by *Agrobacterium* mediated transformation. This was first report of *Agrobacterium* mediated transformation in which axillary buds from 6 months old plants were used as explant. *Agrobacterium* with binary vector pGA492 having *β-glucuronidase*, *neomycin phosphotransferase II* and *bar* genes in between the T-DNA regions was used for transformation. This study proved that phosphinothricin (5.0 mg/L) is more effective selective agent as compared with kanamycin and geneticin. Southern blot analysis was used to confirm the transformants. Leibbrandt and Snyman [7] reported the transformation of *pat* gene in NCo 310 genotype of sugarcane which confers resistance to the herbicide Basta. Stable transgene expression was evaluated in glasshouse and field conditions.

## *4.1.2. Insect resistance*

metabolome (whole metabolites in a specific tissue) has been used as a tool for understanding metabolic regulations. This work was accomplished by some advanced technologies where multiple metabolites were determined in a particular tissue within an hour simultaneously. GCMS (gas chromatography-mass spectrometry) is a vastly used technique that separates the metabolites of different types and identify them on the basis of mass spectral matching and retention time. Identification and extraction methods were optimized for thirty sugarcane metabolites. Hence, metabolome studies are of pivotal importance to understand interaction between the genes and their resultant proteins which can be used to understand mechanisms

Transgenic technology is the only technology through which alien genes may be introduced across the species. Sugarcane has well be explored to engineer for certain valuable agronomic traits and for enhanced sucrose contents. Most of the transformation events reported in sugarcane are through biolistic, anyhow *Agrobacterium* and electroporation have also been used. Success of engineered lines depend upon the integration and stable expression of introduced gene/s. Recalcitrancy, low transformation efficiency, transgene inactivation and difficult backcrossing are major bottlenecks in sugarcane transformation. Though limited reports are available for the field plantation of transgenic sugarcane yet a large number of research groups are

**4.1. Developing genetically modified varieties with improved biotic stress tolerance**

Herbicide resistance is one of the major traits in transgenic plants. It is so desirable that more than 70% of the transgenic crops growing worldwide are herbicide resistant. Various herbicide resistance genes i.e. *EPSPS*, *bar*, *aroA and BXN* have been have been transformed to crop plants for developing herbicide tolerant crops. Crops resistant to glyphosate and glufosinate have been cultivated since 1990s. Plants having ability to tolerate high dose of glyphosate have been developed through biolistic transformation whereas *Agrobacterium* and other methods of transformation (electroporation) have also been tested [59]. Gallo-Meagher and Irvine [60] reported *bar* gene transformation in sugarcane by biolistic gun. Resultant transformants showed tolerance against basta which authenticated effectiveness of *bar* gene not only for herbicide tolerance trait but also as a selectable marker gene, for the selection of putative transformants. Enriquez-Obregon et al. [61] reported transformation of *uid1* and *bar* genes in sugarcane by *Agrobacterium* mediated transformation. They obtained GUS-positive and BASTA resistant calli. Similarly, BASTA resistant variety (Ja 60-5) of sugarcane has also been developed through *Agrobacterium* mediated transformation. Transformation frequencies reached up to 10–35% by employing different transformation protocols. The *PAT* (phosphinothricin acetyltransferase) and *neo* (neomycin phosphotransferase) genes were reported to be transformed in SP80-180 genotype of sugarcane by biolistic

of sucrose accumulation in sugarcane [57].

122 Sugarcane - Technology and Research

involved in engineering sugarcane genome [58].

*4.1.1. Herbicide resistance*

**4. Transgenic approaches for crop improvement**

Insect pests are one of the major yield limiting agents which cause serious losses to crop yield. Economically important insect pests of sugarcane can be categorized into borers, sap sucking pests, white grubs and termites. Sugarcane pests show extensive variation in species composition in different tropic and subtropic agro climatic regions. All around the world, sugarcane is facing problems of insect pests and diseases which are seriously affecting sugar production. No exact estimates are available for these cumulative losses caused by the insect pests and diseases. Anyhow, economic losses caused by certain pests has been estimated. Annual loss of \$10–\$20 million were estimated to sugar industry at Lower Rio Grande Valley of Texas only by *E. loftini*. Similarly, wooly aphid (*Ceratovacuna lanigera*) has been estimated to cause 18.3% yield losses during sixth months [63]. Most of the sugarcane cultivars growing in the field are outcomes of hybridization and selection. Advancements in molecular biology and genetic transformation have helped researchers to develop transgenic sugarcane plants with desired agronomic traits particularly for insect pest resistance. Different types of molecules have been manipulated to produce insect resistant plants such as lectins, proteinase inhibitors, ribosome inactivating proteins, secondary metabolites, delta endotoxins and insecticidal proteins.

Considerable advancements have been made to develop transgenic sugarcane having resistance against lepidopteran borers such as *E. loftini*, *D. saccharalis*, *S. excerptalis* and *C. infuscatellus* by introducing various cry genes. *Bacillus thuringiensis* derived cry genes encoding toxins have been expressed in sugarcane to engineer resistance against insect pests. First transgenic sugarcane was developed by Arencibia [6] against *D. saccharalis*. Five transformation events were selected exhibiting considerable resistance against borer in spite of very low expression (0.59–1.35 ng/mg of soluble leaf protein) of transgene. Truncated *cry1A(b)* gene was expressed in sugarcane under *CaMV 35S* promoter. Lower level of expression was observed in transgenic plants perhaps because of lower activity of the aforementioned promoter in monocots. Low to medium level internode invasions were also observed in the transgenic lines. Transgenic lines were developed with modified GC contents (37.4–47.5%) of *cry1Ac* gene and effect of change in GC contents, was observed on the expression of transgene [69]. Transgene expression was determined as 1–10 and 0.2–6.0 ng/mg of total soluble proteins in the leaves and stem respectively, which was seven times higher than reported by Arencibia et al. [64]. Plants also showed better resistance to sugarcane stem borer, when tested by *in vivo* and *in vitro* insect bioassay. Expression of the transgene was further increased with increase in GC content of *cry1Ac* gene and was determined as 2.2–50 ng/mg of total soluble proteins when GC content was increased to 54.8%. Hence expression of *cry1Ac* gene in sugarcane increased with increase in its GC content [65].

on transgenic plants. Aphid population density was decreased up to 60–80%, and even up to 95% in some lines [69]. Maximum resistance was observed when phloem specific promoter (RSs-1) was used in transformation against sucking pests like wooly aphids. Field performance of transgenic lines is dependent on the expression level and stability of transgene. The most critical factors in this regard are: promoter strength, codon usage, gene silencing and site of integration. Rice polyubiquitin (*RUBIQ2*) promoter has proved as the strongest promoter for transgene expression in sugarcane [70]. Anyhow, maize ubi-1 promoter has also extensively been used in sugarcane for the optimal expression of transgenes. Though maize ubi-1 is a constitutive promoter but it does not give same level of expression in all plant parts, e.g. leaves, roots and stem. Keeping in view the tissue specificity and feeding habits of insect pests, it is necessary to use tissue specific or wound inducible promoters which will overexpress insecticidal proteins only in the target tissues and will prove more effective for the

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**4.2. Developing genetically modified varieties with improved abiotic stress tolerance**

only drought tolerant varieties can be grown with success [71].

Abiotic stresses may alter physiological status of a plant either directly or indirectly by disturbing its metabolism, growth and development. Among abiotic stresses salinity, drought and low temperature are the fundamental factors that significantly influence plant performance. To combat these stresses plant triggers a cascade of physiological and biochemical reactions. Commonly sugars and other osmolytes accumulate in response to abiotic stresses (drought, salinity and low temperature). Water is an essential component for life but its scarcity is increasing day by day throughout the world. Currently almost 65% of fresh water is being used for irrigation, indicating that survival would not be possible without developing drought tolerant varieties in the near future. Sugarcane is a high delta crop and requires heavy irrigation but is now expanding in the regions where water availability is limited, so

Molecular studies have explored that any of the plant growing in stress conditions strives to withstand those conditions by activating certain stress responsive genes/proteins. They include antioxidant enzymes, late embryogenesis abundant proteins, Arabidopsis *HARDY* genes [72], various transcription factors and certain protease inhibitors. Transcriptomic analyses of sugarcane under drought stress has lead to the identification of stress related genes. These genes should be figured out that how critical they are for stress tolerance. Molecular Systems Biology can be used to characterize regulatory networks using model plant. A novel sugarcane gene *Scdr1* (sugarcane drought responsive 1) was overexpressed in tobacco. Its overexpression resulted in increased tolerance against salinity, drought and oxidative stress as was evident by increased photosynthesis, water content, germination rate, biomass, chlorophyll content and reduced accumulation of ROS. Physiological parameters were also less affected as compared to wild type plants. The relationship between anatomical structure and drought tolerance have also been investigated. In the roots of sugarcane, number of vessels per unit area, more veins, widened vesicles in bulliform cells, thick cuticle and less stomata per unit area have close association with drought tolerance [73]. Drought tolerant varieties have better growth of mechanical tissues around the vascular bundle and their thick-walled cells have higher degree of lignification. Various genes that encode transcription factors *bZIP*, *DREB*, *MYB* [74] and

control of insect pests.

Proteinase inhibitors (PIs) derived from both the animals and plants had been introduced in sugarcane to confer resistance against borers. A soybean PI was mixed in the artificial diet of insects and it appeared to have detrimental effects on growth of *D. saccharalis*. Falco and Silva-Filho [66] developed transgenic sugarcane expressing Soybean Kunitz Trypsin Inhibitor (*SKTI*) and Soybean Bowman-Birk Inhibitor (*SBBI*) driven by maize ubi-1 promoter. Transgenic lines were evaluated against *D. saccharalis* by feeding excised leaf tissue and by infecting plants with neonate larvae in the green house. Transgenic lines with *SBBI* did not show significant change in the larvae mortality whereas it was slightly higher when fed on the leaves of *SKTI* expressing plants. Leaves of transgenic plants with *SKTI* or *SBBI* inhibited insect growth and metabolism, hence resulted in reduced insect weight. Dead hearts were also observed in almost all of the plants tested in green house. Without knowing the level of expression, it is very difficult to establish relationship between inhibitor content and dead heart to determine their role in borer resistance. Aportinin appeared to be more effective in inhibting gut proteinases of *S. excerptalis* as compared with those of *C. infuscatellus* and *C. sacchariphagus* indicus. In succeeding studies, aprotinin gene under maize ubi-1 promoter was introduced in sugarcane and transgene expression was determined as 0.16–0.50% of the total soluble leaf proteins. *In vivo* screening assays also revealed very low mortality of *S. excerptalis* larvae but weight of insects was reduced by 99.8% hence insect growth was affected to great extent.

Besides proteinase inhibitors and cry toxins, certain other molecules have also been explored for the effective control of insect pests in sugarcane. Plant derived insecticidal proteins (lectins) are more valuable for the control of insects as compared with bacterial insecticidal proteins. Wheat germ lectin, snowdrop lectin (*Galanthus nivalis* agglutinin, *GNA*) and avidin were used as dietary proteins in bioassays against larvae of sugarcane white grubs. Wheat germ lectins and snowdrop appeared insecticidal and growth inhibitor for *Antitrogus parvulus* larvae. Avidin also appeared to inhibit growth of *A. consanguineus* [67]. Such positive results encouraged researchers to exploit potential of these genes for the control of white grubs. Sétamou et al. [68] included extracts of snowdrop lectin expressing transgenic lines (0.89% in leaf sheath) in the artificial diet (0.47% lectin of total extractable proteins) and examined responses of *D. saccharalis* and *E. loftini* larvae. When artificial diet containing 0.50% of transgenic *GNA* was fed to the larvae of *E. loftini*, a considerable decrease in larvae survival, pupation percentage, adult emergence, pupal weight, longevity and fertility was observed. Transgenic sugarcane expressing lectin under phloem specific RSs-1 (rice sucrose synthase-1) or maize ubi-1 promoters were developed via *Agrobacterium* mediated transformation. Reduced survival rate, development, fertility and feeding was observed in the larvae of *C. lanigera* feeding on transgenic plants. Aphid population density was decreased up to 60–80%, and even up to 95% in some lines [69]. Maximum resistance was observed when phloem specific promoter (RSs-1) was used in transformation against sucking pests like wooly aphids. Field performance of transgenic lines is dependent on the expression level and stability of transgene. The most critical factors in this regard are: promoter strength, codon usage, gene silencing and site of integration. Rice polyubiquitin (*RUBIQ2*) promoter has proved as the strongest promoter for transgene expression in sugarcane [70]. Anyhow, maize ubi-1 promoter has also extensively been used in sugarcane for the optimal expression of transgenes. Though maize ubi-1 is a constitutive promoter but it does not give same level of expression in all plant parts, e.g. leaves, roots and stem. Keeping in view the tissue specificity and feeding habits of insect pests, it is necessary to use tissue specific or wound inducible promoters which will overexpress insecticidal proteins only in the target tissues and will prove more effective for the control of insect pests.

observed on the expression of transgene [69]. Transgene expression was determined as 1–10 and 0.2–6.0 ng/mg of total soluble proteins in the leaves and stem respectively, which was seven times higher than reported by Arencibia et al. [64]. Plants also showed better resistance to sugarcane stem borer, when tested by *in vivo* and *in vitro* insect bioassay. Expression of the transgene was further increased with increase in GC content of *cry1Ac* gene and was determined as 2.2–50 ng/mg of total soluble proteins when GC content was increased to 54.8%. Hence expression of *cry1Ac* gene in sugarcane increased with increase

Proteinase inhibitors (PIs) derived from both the animals and plants had been introduced in sugarcane to confer resistance against borers. A soybean PI was mixed in the artificial diet of insects and it appeared to have detrimental effects on growth of *D. saccharalis*. Falco and Silva-Filho [66] developed transgenic sugarcane expressing Soybean Kunitz Trypsin Inhibitor (*SKTI*) and Soybean Bowman-Birk Inhibitor (*SBBI*) driven by maize ubi-1 promoter. Transgenic lines were evaluated against *D. saccharalis* by feeding excised leaf tissue and by infecting plants with neonate larvae in the green house. Transgenic lines with *SBBI* did not show significant change in the larvae mortality whereas it was slightly higher when fed on the leaves of *SKTI* expressing plants. Leaves of transgenic plants with *SKTI* or *SBBI* inhibited insect growth and metabolism, hence resulted in reduced insect weight. Dead hearts were also observed in almost all of the plants tested in green house. Without knowing the level of expression, it is very difficult to establish relationship between inhibitor content and dead heart to determine their role in borer resistance. Aportinin appeared to be more effective in inhibting gut proteinases of *S. excerptalis* as compared with those of *C. infuscatellus* and *C. sacchariphagus* indicus. In succeeding studies, aprotinin gene under maize ubi-1 promoter was introduced in sugarcane and transgene expression was determined as 0.16–0.50% of the total soluble leaf proteins. *In vivo* screening assays also revealed very low mortality of *S. excerptalis* larvae but weight of insects was reduced by 99.8% hence insect growth was

Besides proteinase inhibitors and cry toxins, certain other molecules have also been explored for the effective control of insect pests in sugarcane. Plant derived insecticidal proteins (lectins) are more valuable for the control of insects as compared with bacterial insecticidal proteins. Wheat germ lectin, snowdrop lectin (*Galanthus nivalis* agglutinin, *GNA*) and avidin were used as dietary proteins in bioassays against larvae of sugarcane white grubs. Wheat germ lectins and snowdrop appeared insecticidal and growth inhibitor for *Antitrogus parvulus* larvae. Avidin also appeared to inhibit growth of *A. consanguineus* [67]. Such positive results encouraged researchers to exploit potential of these genes for the control of white grubs. Sétamou et al. [68] included extracts of snowdrop lectin expressing transgenic lines (0.89% in leaf sheath) in the artificial diet (0.47% lectin of total extractable proteins) and examined responses of *D. saccharalis* and *E. loftini* larvae. When artificial diet containing 0.50% of transgenic *GNA* was fed to the larvae of *E. loftini*, a considerable decrease in larvae survival, pupation percentage, adult emergence, pupal weight, longevity and fertility was observed. Transgenic sugarcane expressing lectin under phloem specific RSs-1 (rice sucrose synthase-1) or maize ubi-1 promoters were developed via *Agrobacterium* mediated transformation. Reduced survival rate, development, fertility and feeding was observed in the larvae of *C. lanigera* feeding

in its GC content [65].

124 Sugarcane - Technology and Research

affected to great extent.

#### **4.2. Developing genetically modified varieties with improved abiotic stress tolerance**

Abiotic stresses may alter physiological status of a plant either directly or indirectly by disturbing its metabolism, growth and development. Among abiotic stresses salinity, drought and low temperature are the fundamental factors that significantly influence plant performance. To combat these stresses plant triggers a cascade of physiological and biochemical reactions. Commonly sugars and other osmolytes accumulate in response to abiotic stresses (drought, salinity and low temperature). Water is an essential component for life but its scarcity is increasing day by day throughout the world. Currently almost 65% of fresh water is being used for irrigation, indicating that survival would not be possible without developing drought tolerant varieties in the near future. Sugarcane is a high delta crop and requires heavy irrigation but is now expanding in the regions where water availability is limited, so only drought tolerant varieties can be grown with success [71].

Molecular studies have explored that any of the plant growing in stress conditions strives to withstand those conditions by activating certain stress responsive genes/proteins. They include antioxidant enzymes, late embryogenesis abundant proteins, Arabidopsis *HARDY* genes [72], various transcription factors and certain protease inhibitors. Transcriptomic analyses of sugarcane under drought stress has lead to the identification of stress related genes. These genes should be figured out that how critical they are for stress tolerance. Molecular Systems Biology can be used to characterize regulatory networks using model plant. A novel sugarcane gene *Scdr1* (sugarcane drought responsive 1) was overexpressed in tobacco. Its overexpression resulted in increased tolerance against salinity, drought and oxidative stress as was evident by increased photosynthesis, water content, germination rate, biomass, chlorophyll content and reduced accumulation of ROS. Physiological parameters were also less affected as compared to wild type plants. The relationship between anatomical structure and drought tolerance have also been investigated. In the roots of sugarcane, number of vessels per unit area, more veins, widened vesicles in bulliform cells, thick cuticle and less stomata per unit area have close association with drought tolerance [73]. Drought tolerant varieties have better growth of mechanical tissues around the vascular bundle and their thick-walled cells have higher degree of lignification. Various genes that encode transcription factors *bZIP*, *DREB*, *MYB* [74] and *RD26* have also been evaluated to enhance stress tolerance in plants. Transcription factors of *MYB* superfamily which are in abundant, also play crucial role in growth and development under stress conditions [75]. Expression profiling of sugarcane was performed under stress conditions to identify abiotic-stress-inducible genes. Wild type Q117 sugarcane plants were exposed to salinity, drought and cold stresses. Variations in the expression level of four genes encoding for galactinol synthase (*GolS*), late embryogenesis abundant protein 3 (*LEA3*), early response to dehydration protein 4 (*ERD4*) and pyrroline-5-carboxylase synthetase (*P5CS*) were evaluated by real-time PCR. *P5CS* and *GolS* were strongly induced under salt stress whereas, *LEA3* and *ERD4* were induced under cold and drought stress respectively. Overexpression of *CBF4* (C-repeat binding factor 4) gene from *Arabidopsis thaliana* in Q117 led to a considerable increase in the expression of *P5CS* and *ERD4* as compared with wild-type plants under normal conditions. Anyhow expression of *LEA3* and *GolS* did not appear to be affected in transgenic plants. These results suggested presence of active abiotic stress-inducible genes in sugarcane and that expression of *Arabidopsis CBF4* gene in sugarcane can activate stress tolerance genes under normal conditions [76].

many studies have been reported on the genes involved in sucrose metabolism directly or indirectly. SPS (sucrose phosphate synthase), SPP (sucrose phosphate phosphatase), SuSy (sucrose synthase), HK (hexokinase), FK (fructokinase) VAI (vacuolar acid invertase), NAI (neutral acid invertase), CWI (cell wall invertase), SAI (soluble acid invertase), PFK (ATP dependent phosphofructokinase), PFP (pyrophosphate dependent phosphofructokinase), UDPase (UDP glucose pyrophosphorylase), ADP-G-PP- (ADP-Glucose pyrophosphorylase) and sucrose transporters (SUT1 and SUT4) are the key enzymes involved in sucrose metabolic pathway [79]. Engineering these enzymes through genetic transformation may lead to

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Fructose 6-phosphate 1-phosphotransferase catalyzes the principal reaction of glycolysis i.e. the reversible conversion of pyrophosphate (PPi) and fructose 6-phosphate (Fru 6-P) into inorganic phosphate (Pi) and fructose 1,6-bisphosphate (Fru 1,6-P2). Pyrophosphate

**Figure 2.** Schematic sketch showing the most critical enzymes involved in sucrose accumulation in sugarcane culmn.

increased sucrose accumulation and of course sugar recovery (**Figure 2**).

Plants use complex mechanisms to adapt ionic/osmotic stresses and accumulate compatible solutes to cope with these stress conditions. Salt stress has drastic effects on photosynthetic activity which affects crop production, product quality and of course sucrose accumulation in cane stalk. These stresses disturb homeostasis at cellular level and even at plant level. It is very critical for the plant to maintain low level of toxic ions in the cell but under salt stress, Na<sup>+</sup> and Cl− ions accumulate in the cytoplasm due to their inability to pump them out. The level of Na<sup>+</sup> ion is regulated by specific transporters i.e. plasma membrane Na<sup>+</sup> /H<sup>+</sup> antiporter *SOS1*. When *AVP1* (Arabidopsis Vacuolar Pyrophosphatase) gene was overexpressed in sugarcane by Kumar et al. [77], increased tolerance against drought and salinity was observed in the transgenic plants. Profused and longer roots were observed in transgenic plants as compared to control. Concurrently, survival of transgenic plants under drought and salt stress indicated their increased level of tolerance against drought and salinity. Constitutive expression of *AVP1* gene improved plant growth under different abiotic stresses. Many explanations have been anticipated including better vacuolar ion sequestration, enhanced auxin transport, increased heterotrophic growth, and more sucrose transport from source to sink tissues. Mutant plants which lack functional *AVP1* gene and transgenic plants for *AVP1* were used to evaluate its role. It becomes clear that AVP1 is a protein with multiple functions. Systems Biology can be of great help for the complete understanding of these complex biological networks [78].

#### **4.3. Developing genetically modified varieties with improved sugar recovery**

Sugarcane has capacity to store more than 25% sucrose of its fresh weight, so a great potential is there to increase sugar recovery. Advancements in biotechnological tools has helped to understand metabolic pathways involved in sucrose accumulation in sugarcane. Enzymes and control points involved in sucrose metabolic pathway, photosynthetic efficiency, degree of phloem loading/unloading, rate of sucrose assimilation and carbon partitioning within the stem and vacuoles are the key targets which needs to be explored for increasing sucrose accumulation. Expression analysis of genes in relation to sucrose accumulation can be of great help to understand role of various genes involved in sucrose metabolism. Until now many studies have been reported on the genes involved in sucrose metabolism directly or indirectly. SPS (sucrose phosphate synthase), SPP (sucrose phosphate phosphatase), SuSy (sucrose synthase), HK (hexokinase), FK (fructokinase) VAI (vacuolar acid invertase), NAI (neutral acid invertase), CWI (cell wall invertase), SAI (soluble acid invertase), PFK (ATP dependent phosphofructokinase), PFP (pyrophosphate dependent phosphofructokinase), UDPase (UDP glucose pyrophosphorylase), ADP-G-PP- (ADP-Glucose pyrophosphorylase) and sucrose transporters (SUT1 and SUT4) are the key enzymes involved in sucrose metabolic pathway [79]. Engineering these enzymes through genetic transformation may lead to increased sucrose accumulation and of course sugar recovery (**Figure 2**).

*RD26* have also been evaluated to enhance stress tolerance in plants. Transcription factors of *MYB* superfamily which are in abundant, also play crucial role in growth and development under stress conditions [75]. Expression profiling of sugarcane was performed under stress conditions to identify abiotic-stress-inducible genes. Wild type Q117 sugarcane plants were exposed to salinity, drought and cold stresses. Variations in the expression level of four genes encoding for galactinol synthase (*GolS*), late embryogenesis abundant protein 3 (*LEA3*), early response to dehydration protein 4 (*ERD4*) and pyrroline-5-carboxylase synthetase (*P5CS*) were evaluated by real-time PCR. *P5CS* and *GolS* were strongly induced under salt stress whereas, *LEA3* and *ERD4* were induced under cold and drought stress respectively. Overexpression of *CBF4* (C-repeat binding factor 4) gene from *Arabidopsis thaliana* in Q117 led to a considerable increase in the expression of *P5CS* and *ERD4* as compared with wild-type plants under normal conditions. Anyhow expression of *LEA3* and *GolS* did not appear to be affected in transgenic plants. These results suggested presence of active abiotic stress-inducible genes in sugarcane and that expression of *Arabidopsis CBF4* gene in sugarcane can activate stress tolerance genes

Plants use complex mechanisms to adapt ionic/osmotic stresses and accumulate compatible solutes to cope with these stress conditions. Salt stress has drastic effects on photosynthetic activity which affects crop production, product quality and of course sucrose accumulation in cane stalk. These stresses disturb homeostasis at cellular level and even at plant level. It is very critical for the plant to maintain low level of toxic ions in the cell but under salt stress, Na<sup>+</sup>

When *AVP1* (Arabidopsis Vacuolar Pyrophosphatase) gene was overexpressed in sugarcane by Kumar et al. [77], increased tolerance against drought and salinity was observed in the transgenic plants. Profused and longer roots were observed in transgenic plants as compared to control. Concurrently, survival of transgenic plants under drought and salt stress indicated their increased level of tolerance against drought and salinity. Constitutive expression of *AVP1* gene improved plant growth under different abiotic stresses. Many explanations have been anticipated including better vacuolar ion sequestration, enhanced auxin transport, increased heterotrophic growth, and more sucrose transport from source to sink tissues. Mutant plants which lack functional *AVP1* gene and transgenic plants for *AVP1* were used to evaluate its role. It becomes clear that AVP1 is a protein with multiple functions. Systems Biology can be of great help for the complete understanding of these complex biological networks [78].

ion is regulated by specific transporters i.e. plasma membrane Na<sup>+</sup>

**4.3. Developing genetically modified varieties with improved sugar recovery**

Sugarcane has capacity to store more than 25% sucrose of its fresh weight, so a great potential is there to increase sugar recovery. Advancements in biotechnological tools has helped to understand metabolic pathways involved in sucrose accumulation in sugarcane. Enzymes and control points involved in sucrose metabolic pathway, photosynthetic efficiency, degree of phloem loading/unloading, rate of sucrose assimilation and carbon partitioning within the stem and vacuoles are the key targets which needs to be explored for increasing sucrose accumulation. Expression analysis of genes in relation to sucrose accumulation can be of great help to understand role of various genes involved in sucrose metabolism. Until now

ions accumulate in the cytoplasm due to their inability to pump them out. The level

/H<sup>+</sup>

antiporter *SOS1*.

under normal conditions [76].

126 Sugarcane - Technology and Research

and Cl−

of Na<sup>+</sup>

Fructose 6-phosphate 1-phosphotransferase catalyzes the principal reaction of glycolysis i.e. the reversible conversion of pyrophosphate (PPi) and fructose 6-phosphate (Fru 6-P) into inorganic phosphate (Pi) and fructose 1,6-bisphosphate (Fru 1,6-P2). Pyrophosphate

**Figure 2.** Schematic sketch showing the most critical enzymes involved in sucrose accumulation in sugarcane culmn.

dependent phosphofructokinase (PFP) is also partially responsible for being cycled between the hexose phosphate and triose phosphate pools. This cycling was downregulated by constitutive expression of untranslatable and antisense forms of *PFP-b* gene. Approximately 70% activity was decreased in young internodal tissues and no activity was observed in mature tissues. Hendrik and Botha [80] reported decrease in sugar yield as the result of decrease in the PFP activity. A significant increase in sucrose content (in more than 50% of the lines) was observed in the immature internodes, but even 30% downregulation of Pyrophosphate dependent phosphofructokinase (PFP) activity did not affect the mature internodes as compared to wild type. Mature internodes of most of the transgenic lines showed higher sucrose accumulation but was not significant. Hence Pyrophosphate dependent phosphofructokinase (PFP) activity in internodal tissues of sugarcane has a positive relation with respiration and is inversely related with sucrose content. In transgenic plants, no significant difference was observed in development and growth of plants both under greenhouse and field conditions. So PFP (pyrophosphate dependent phosphofructokinase) influences the sucrose accumulation ability of biosynthetically active and young culm of sugarcane. Equilibrium of glycolytic intermediates (stored sucrose) is restored when ATP dependent phosphofructokinase and the PFP activity is sufficient.

increase in isomaltulose (IM) whereas, vacuole-targeted expression of transgene resulted in significant increase in the isomaltulose (IM) accumulation under greenhouse conditions [91]. Hamerli and Birch [84] reported the first field trial of transgenic sugarcane producing trehalulose (TH). Synthetic sweeteners, an alternative to sucrose are produced through fermentation or chemical reactions which are very expensive. Production of sweeteners in sugarcane through targeted expression of transgene in the mature stem can be an economical alternative. For targeted delivery of proteins into the plastids and vacuoles, transit peptides have already been established in transformants. Zhang et al. [85] worked out not only to develop abiotic stress tolerant sugarcane but also on engineering metabolic pathways for improved trehalose (a valuable sugar moiety) content. Directing sucrose accumulation to vacuole in spite of cytosol may prove an effective strategy for enhanced sucrose accumulation because vacuole occupies large space in the cell. Hence, biotechnological interventions can do a lot to improve sugar recovery in this sweet grass.

Biotechnological Interventions for the Improvement of Sugarcane Crop and Sugar Production

http://dx.doi.org/10.5772/intechopen.71496

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**5. Potential of biotechnology to promote sugarcane as a future** 

Plant biomass from grasses including sugarcane, can be used as a renewable source of energy by converting their cellulose, hemicellulose and lignin into bioethanol. Plant derived biofuels reduce dependence on fossil fuels and is of great importance in the countries where oil reserves are limited. Engineering plastid genome of sugarcane may prove a great milestone in this regard [86]. Biofuels produced from plant lignocellulosic biomass (second generation biomass) have advantage over first generation biomass in term of CO2 balance and net energy. Another advantage is that they have no competition for supplies with food industries. As a result, production of bioethanol from 2nd generation biomass is more economical. Sugarcane is one of the most economical source of bioethanol all over the world. Brazil is the leading country in this regard and 50% of the country energy needs are fulfilled by sugarcane ethanol [87]. National fuel alcohol program (ProAlcooL) was launched by Brazil. Major aim of this program was to replace usage of gasoline with bioethanol. In Brazil, 6.19 billion gallons (23.4 billion liters) of ethanol was produced from sugarcane whereas 15% of the total electricity was generated from sugarcane bagasse during 2014 [88]. The genetic foundation of current sugarcane breeding program started with interspecific hybrid varieties originated from early breeding activities in West Indies, India (e.g. Co 206, Co 207) and Indonesia (e.g. POJ 2878, POJ 2364). Sugar yield was increased up to 1–2% per annum by sugarcane programs and most of this increase is attributed to genetics. Conventional breeding in sugarcane has certain limits, as a result desired results could not be achieved. *Saccharum* spp. is genetically complex having 2n = 100–130 with intricate genomic makeup evolved through highly successful interspecific hybridization between *Saccharum spontaneum* and *Saccharum officinarum*, which have well been explored to develop commercial varieties. Ming et al. [89] summarized usage of conventional and molecular approaches for the genetic improvement of sugarcane making it world's most efficient crop in terms of conversion of solar energy into chemical energy.

In sugarcane, about two-third of the photosynthetically fixed carbon is stored in the form of cellulose and hemicellulose. Sugarcane mills produce millions of tons of bagasse annually

**energy crop**

Sugarcane culm is an important experimental system to elucidate biochemical and molecular mechanisms involved in sucrose accumulation or carbon partitioning for the application of gene expression studies in this context [81]. Vacuolar targeted expression of sucrose isomerase gene doubled sucrose accumulation in the culm of greenhouse growing plants. Engineered sugarcane plants not only depicted enhanced sucrose transport but also photosynthesis and sink strengths were improved. These results highlighted importance of sugarcane as an energy crop as more carbon source would result in more biofuel production. Higher level of sucrose and accumulation of isomaltulose (a high value sugar) has also been reported in sugarcane [82]. An experimental study was conducted to explore biosynthesis of isomaltulose (IM) through engineering metabolic pathways. Sucrose (α-D- glucopyranosyl 1,2-D- fructofuranose) is converted into isomaltulose (α-D-glucopyranosyl-1,6-D-fructofuranose) by some bacteria. This sucrose is resistant to several microorganisms as is not metabolized by invertases. Easy digestion (likewise glucose and fructose) by humans is another significant advantage of this sweetener. Instead of salivary invertases, intestinal disaccharidase is involved in the digestion of isomaltulose, so its digestion is relatively slow. Anyhow, it is beneficial because it does not affect insulin and blood glucose levels. Owing to be acariogenic, non-hygroscopic, stable and slowly digestible sweetener, it has mounting market. Biosynthesis of isomaltulose (IM) involves a sucrose isomerase (SI) that does not require cofactor and substrate activation [83]. More isomaltulose (IM) is produced in sugarcane culmn when highly efficient sucrose isomerase (SI) is targeted to vacuole. Further, IM (isomaltulose) could be accumulated without any prominent decrease in sucrose content. Sucrose contents appeared to be doubled in selected transgenic lines but further studies would be required for commercial scale application of this trait i.e. patterns of developmental expression, compartmentation and enzyme stability resulting in high isomaltulose (IM) content. Hence, sucrose isomers can be produced in sugarcane by transgenic technology. Isomaltulose was produced either by expressing sucrose isomerase in the apoplast or in the vacuole. Apoplast-targeted expression did not show any significant increase in isomaltulose (IM) whereas, vacuole-targeted expression of transgene resulted in significant increase in the isomaltulose (IM) accumulation under greenhouse conditions [91]. Hamerli and Birch [84] reported the first field trial of transgenic sugarcane producing trehalulose (TH). Synthetic sweeteners, an alternative to sucrose are produced through fermentation or chemical reactions which are very expensive. Production of sweeteners in sugarcane through targeted expression of transgene in the mature stem can be an economical alternative. For targeted delivery of proteins into the plastids and vacuoles, transit peptides have already been established in transformants. Zhang et al. [85] worked out not only to develop abiotic stress tolerant sugarcane but also on engineering metabolic pathways for improved trehalose (a valuable sugar moiety) content. Directing sucrose accumulation to vacuole in spite of cytosol may prove an effective strategy for enhanced sucrose accumulation because vacuole occupies large space in the cell. Hence, biotechnological interventions can do a lot to improve sugar recovery in this sweet grass.
